The invention provides new materials and a novel method for their preparation by incorporating molecular, nanoscale, and macroscopic materials within a cellulose matrix. The process involves encapsulation or immobilization of the active solid substance in a cellulose framework by regenerating cellulose dissolved in an ionic liquid solvent in a regenerating solution. The active substance can be initially present in the ionic liquid, or in the regenerating solvent, either as a solution, or as a dispersion. The invention is applicable to molecular encapsulation and to entrapping of larger particles including enzymes, nanoparticles and macroscopic components, and to the formation of bulk materials with a wide range of morphological forms.
The use of ionic liquids as replacements for conventional organic solvents in chemical, biochemical and separation processes has been demonstrated. Graenacher first suggested a process for the preparation of cellulose solutions by heating cellulose in a liquid N-alkylpyridinium or N-arylpyridinium chloride salt, U.S. Pat. No. 1,943,176, especially in the presence of a nitrogen-containing base such as pyridine. However, that finding seems to have been treated as a novelty of little practical value because the molten salt system was, at the time, somewhat esoteric. This original work was undertaken at a time when ionic liquids were essentially unknown and the application and value of ionic liquids as a class of solvents had not been realized.
Ionic liquids are now a well-established class of liquids containing solely ionized species, and having melting points largely below 150xc2x0 C. or most preferably 100xc2x0 C. In most cases, ionic liquids (IL) are organic salts containing one or more cations that are typically ammonium, imidazolium or pyridinium ions, although many other types are known. The range of ionic liquids that are applicable to the dissolution of cellulose are disclosed in U.S. patent application Ser. No. 10/256,521, filed on Sep. 27, 2002, entitled xe2x80x9cDissolution and Processing of Cellulose Using Ionic Liquidsxe2x80x9d, that claimed priority from provisional application Serial No. 60/326,704 that was filed on Oct. 3, 2001, and in Swatloski et al., J. Am. Chem. Soc. 2002, 124:4974-4975.
Traditional cellulose dissolution processes, including the cuprammonium and xanthate processes, are often cumbersome or expensive and require the use of unusual solvents, typically with a high ionic strength and are used under relatively harsh conditions. [Kirk-Othmer xe2x80x9cEncyclopedia of Chemical Technologyxe2x80x9d, Fourth Edition 1993, volume 5, p. 476-563.] Such solvents include carbon disulfide, N-methylmorpholine-N-oxide (NMMNO), mixtures of N,N-dimethylacetamide and lithium chloride (DMAC/LiCl), dimethylimidazolone/LiCl, concentrated aqueous inorganic salt solutions [ZnCl/H2O, Ca(SCN)2/H2O], concentrated mineral acids (H2SO4/H3PO4) or molten salt hydrates (LiClO4.3H2O, NaSCN/KSCN/LiSCN/H2O).
These traditional cellulose dissolution processes break the cellulose polymer backbone resulting in regenerated products that contain an average of about 500 to about 600 glucose units per molecule rather than the native larger number of about 1500 or more glucose units per molecule. In addition, processes such as that used in rayon formation proceed via xanthate intermediates and tend to leave some residual derivatized (substituent groups bonded to) glucose residues as in xanthate group-containing cellulose.
For example, U.S. Pat. No. 5,792,399 teaches the use of N-methylmorpholine-N-oxide (NMMNO) solutions of cellulose to prepare regenerated cellulose that contained polyethyleneimine (PEI). That patent teaches that one should utilize a pre-treatment with the enzyme cellulase to lessen the molecular weight to the cellulose prior to dissolution. In addition, it is taught that NMMNO decomposes at the temperatures used for dissolution to provide N-methylmorpholine as a degradation product that could be steam distilled away from the cellulose solution. The presence of PEI is said to lessen the decomposition of the NMMNO.
Other traditional processes that can provide a solubilized cellulose do so by forming a substituent that is intended to remain bonded to the cellulose such as where cellulose esters like the acetate and butyrate esters are prepared, or where a carboxymethyl, methyl, ethyl, C2-C3 2-hydroxyalkyl (hydroxyethyl or hydroxypropyl), or the like group is added to the cellulose polymer. Such derivative (substituent) formation also usually leads to a lessening of the degree of cellulose polymerization so that the resulting product contains fewer cellobiose units per molecule than the cellulose from which it was prepared.
Thus, Linko and co-worker reported dissolving relatively low molecular weight cellulose (DP=880) in a mixture of N-ethylpyridinium chloride (NEPC) and dimethylformamide, followed by cooling to 30xc2x0 C., incorporation of various microbial cells into the solution and then regeneration of the cellulose into a solid form by admixture with water. [Linko et al., Enzyme Microb. Technol., 1:26-30 (1979).] That research group also reported entrapment of yeast cells in a solution of 1 percent cellulose dissolved in a mixture of NEPC and dimethyl sulfoxide, as well as entrapment using 7.5 to 15 percent cellulose di- or triacetates dissolved in several organic solvents. [Weckstrom et al., in Food Engineering in Food Processing, Vol. 2, Applied Science Publishers Ltd., pages 148-151 (1979).]
Entrapped materials have a wide number of uses, from controlled release systems to structural modifiers and sensor or reactive materials. The entrapped materials can be formulated as membranes, coatings or capsules. Methods are known for forming encapsulated products including emulsion polymerization, interfacial polymerization, desolution, emulsification, gelation, spray-drying, vacuum coating, and adsorption onto porous particles. Common materials used include polymers, hydrocolloids, sugars, waxes, fats, metals and metal oxides.
The use of membranes, coatings, and capsules for the controlled release of liquid materials is well known in the art of both agricultural and non-agricultural chemicals, including the preparation of graphic arts materials, pharmaceuticals, food, and pesticide formulations. In agriculture, controlled-release techniques have improved the efficiency of herbicides, insecticides, fungicides, bactericides, and fertilizers. Non-agricultural uses include encapsulated dyes, inks, pharmaceuticals, flavoring agents, and fragrances.
The most common forms of controlled-release materials are coated droplets or microcapsules, coated solids, including both porous and non-porous particles, and coated aggregates of solid particles. In some instances, a water-soluble encapsulating film is desired, which releases the encapsulated material when the capsule is placed in contact with water. Other coatings are designed to release the entrapped material when the capsule is ruptured or degraded by external force. Still further coatings are porous in nature and release the entrapped material to the surrounding medium at a slow rate by diffusion through the pores.
Materials have been formulated as emulsifiable concentrates by dissolving the materials in an organic solvent mixed with a surface-active agent or as an oily agent. In solid form, the insecticides have been formulated as a wettable powder in which the insecticide is adsorbed onto finely powdered mineral matter or diatomaceous earth, as a dust or as granules.
However, these conventional formulations pose a variety of problems such as the pollution of the environment caused by the organic solvent used in the emulsions or by the dust resulting from the wettable powders. Furthermore, for these formulations to have long-term residual effectiveness, an amount much higher than that used in normal application is required, and this increased amount can affect the environment or cause problems of safety. Other conventional microcapsules that encapsulate active insecticidal components are obtained through an interfacial polymerization reaction and are not ideal in terms of the production process or as an effective stabilized insecticide.
There is therefore a strong demand for a formulation that maintains a high degree of efficacy over long periods. Given this background, research and development are now actively under way to develop a superior microencapsulated formulation that can effectively replace the emulsifiable concentrate, interfacially-polymerized or wettable powders, and is safer to use.
Enzyme entrapment on solid supports is a well-established technique for improving stability and separations aspects in enzymatic transformations. Entrapment of enzymes on solid supports can result in improved stability to pH and temperature and aid in separation of the enzyme from the reaction mixture, and also for formation of enzyme electrodes for sensor applications.
There are four principal methods available for immobilizing enzymes: adsorption, covalent binding, entrapment, and membrane confinement. A common method for immobilization is to use polysaccharide activation in which cellulose beads are reacted under alkali conditions with cyanogen bromide. The intermediate produced is then covalently coupled with soluble enzymes. Examples are lactase, penicillin acylase, and aminoacylase enzymes.
Entrapment of enzymes within gels or fibers is a convenient method for use in processes involving low molecular weight substrates and products. Entrapment is the method of choice for immobilization of microbial, animal and plant cells. Calcium alginate is widely used. Enzymes can be entrapped in cellulose acetate fibers by formulation of an emulsion of the enzyme plus cellulose acetate in dichloromethane, followed by extrusion of fibers.
Entrapped enzyme methods have wide applicability, but the entrapped enzymes can be technically difficult to prepare and involve moderate to high costs. Hence, new methods of preparing entrapped enzymes are desirable.
The disclosure hereinafter describes the preparation of encapsulated materials in a cellulose matrix by dispersion and regeneration of IL/cellulose solutions containing an active substance into a regenerating liquid in which the IL is soluble and that is a non-solvent for cellulose and the active agent. It will be clear to those skilled in the art that this invention is applicable to the formulation of beads and fibers in which active agents are entrapped.
The present invention contemplates a cellulose-encapsulated active substance and an encapsulation method for active substances to form a regenerated cellulose matrix in which the active substance is distributed throughout the matrix. The distribution of the active substance is preferably substantially homogeneous within the matrix of regenerated cellulose. The regenerated cellulose (i) has about the same molecular weight as the original cellulose from which it is prepared and typically a degree of polymerization (DP) of about 1200, and (ii) is substantially free of substituent groups and entrapped ionic liquid degradation products. The material to be encapsulated (active substance) is dispersed, preferably homogeneously, or dissolved in a hydrophilic ionic liquid that is substantially free of water, an organic solvent or nitrogen-containing base containing solubilized cellulose, and the cellulose is subsequently reformed (regenerated) as a solid in which the active substance is dispersed in the cellulose matrix, preferably homogeneously.
This method has advantages for formation of composites containing many solid substances which are desirable to encapsulate in a cellulose matrix, particularly for the incorporation of active agents that are not soluble in water or other common solvents, for example nanoparticles or macroscopic materials.
Matrices formed by this process are capable of effecting a slow rate of release of the encapsulated materials by diffusion through the shell to the surrounding medium, swelling in a liquid medium such as water, by slow, controlled degradation of the cellulose matrix structure, or by slow dissolution of the active substance from within the matrix.
Materials suitable for encapsulation include chemical-biological agents such as herbicides, insecticides, fungicides, bactericides, animal, insect, and bird repellent, plant growth regulators, fertilizers, and flavor and odor compositions, catalysts, photoactive agents, indicators, dyes, and UV adsorbents.
The final morphological form of the encapsulated composite depends on the regeneration process and on the desired applications of the materials. For example, high surface area beads, cylinders or flocs can be manufactured for filtration or separation applications, whereas thin films can be prepared for membrane and sensor uses.
Entrapment of biomolecules on solid supports is a well-established technique for improving pH and temperature stability particularly for enzymes and whole cells. Entrapment of biomolecules within a cellulose support can result in new materials for sensing and detection application.
Macroscopic magnetite particles can be incorporated into cellulose to prepare magnetically modified materials. These materials have a number of applications in magnetic fluidized bed extraction processes for protein and metal extraction or detection.